The limited tryptic cleavage of chymotryptic S-1 : An approach to the characterization of the actin site in myosin heads

Mornet, Dominique; Pantel, Pierre; Audemard, Etienne; Kassab, Rhida

doi:10.1016/0006-291x(79)91867-9

Cited by 205 publications

(82 citation statements)

References 23 publications

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“…11). The typical digestion pattern of the acto-S1 complex described by Mornet et al [6], i.e. the inhibition of splitting at the 50-kDa/20-kDa junction, can be observed in Fig.…”

Section: Effect Of Nucleotides On the Fragmentation Of Acto-si Complexesmentioning

confidence: 71%

Effect of nucleotides, divalent cations and temperature on the tryptic susceptibility of myosin subfragment 1

Mócz

Szilágyi

et al. 1984

European Journal of Biochemistry

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The kinetics of tryptic breakdown of the heavy chain of chymotryptic myosin subfragment 1 (Sl) according to the following scheme (where the numbers respresent approximate masses in kDa) are altered at 21 "C by divalent process affects the tryptic fragmentation of S1 similarly to, but less effectively than, nucleotides. Acts-S1 formation prevents the effect of ATP on fragmentation. At 37 'C S1 loses ATPase activity; tryptic digestion proceeds more rapidly and the 50-kDa and 25-kDa fragments are degraded to small peptides. Nucleotides protect against the effects of higher temperature by producing conformational changes not only in the 27-kDa N-terminal portion (containing the putative nucleotide binding site) of the heavy chain of S1 but also in the 50-kDa peptide.Previous studies have shown that, on further digestion with trypsin, the heavy chain of chymotryptic myosin subfragment 1 (Sl) is split into two fragments with approximate masses of 75 kDa and 20kDa; the latter contains the two reactive thiols, SH-1 and SH-2 [I]. The 75-kDa fragment is further degraded into an N-terminal 25-kDa fragment and an adjacent 50-kDa polypeptide with the transient appearance of a 27-kDa fragment [I -31. It has been suggested that the 25-kDa fragment, which was found to be the binding site of a photoaffinity ATP analog [4], is formed by two parallel routes directly from the 75-kDa fragment and indirectly with the 27-kDa fragment as precursor [ 5 ] .Alterations in the proteolytic fragmentation pattern induced by actin, nucleotide or metal binding indicate that structural changes take place in the myosin head region upon their interactions. Thus actin inhibits the proteolytic cleavage of the S1 heavy chain at the junction between the 50-kDa and 20-kDa fragments. The cleavage abolishes the A preliminary report has been presented [(1982) Biophys. J . 37, 38al.Abbreviations. AdoPP[NH]P, adenosine 5'-[P,y-imido]triphosphate; HMM, heavy meromyosin; S1, myosin subfragment 1 ; Nbs,, 5,5'-dithiobis(2-nitrobenzoic acid) ; NaDodSO,, sodium dodcecyl sulfate.

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“…11). The typical digestion pattern of the acto-S1 complex described by Mornet et al [6], i.e. the inhibition of splitting at the 50-kDa/20-kDa junction, can be observed in Fig.…”

Section: Effect Of Nucleotides On the Fragmentation Of Acto-si Complexesmentioning

confidence: 71%

Effect of nucleotides, divalent cations and temperature on the tryptic susceptibility of myosin subfragment 1

Mócz

Szilágyi

et al. 1984

European Journal of Biochemistry

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“…In this study, we focused our attention on loops 2 and 3 of Chara myosin. Loops 2 and 3 are part of actin binding sites (9,10). Cross-linking studies suggested that positively charged residues in loops 2 and 3 interact with negatively charged residues in subdomain 1 of actin at the initial weakly bound state (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21).…”

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confidence: 99%

Unique charge distribution in surface loops confers high velocity on the fast motor protein Chara myosin

Ito

Yamaguchi

Yanase

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Most myosins have a positively charged loop 2 with a cluster of lysine residues that bind to the negatively charged N-terminal segment of actin. However, the net charge of loop 2 of very fast Chara myosin is zero and there is no lysine cluster in it. In contrast, Chara myosin has a highly positively charged loop 3. To elucidate the role of these unique surface loops of Chara myosin in its high velocity and high actin-activated ATPase activity, we have undertaken mutational analysis using recombinant Chara myosin motor domain. It was found that net positive charge in loop 3 affected Vmax and Kapp of actin activated ATPase activity, while it affected the velocity only slightly. The net positive charge in loop 2 affected Kapp and the velocity, although it did not affect Vmax. Our results suggested that Chara myosin has evolved to have highly positively charged loop 3 for its high ATPase activity and have less positively charged loop 2 for its high velocity. Since high positive charge in loop 3 and low positive charge in loop 2 seem to be one of the reasons for Chara myosin's high velocity, we manipulated charge contents in loops 2 and 3 of Dictyostelium myosin (class II). Removing positive charge from loop 2 and adding positive charge to loop 3 of Dictyostelium myosin made its velocity higher than that of the wild type, suggesting that the charge strategy in loops 2 and 3 is widely applicable.actin ͉ ATPase ͉ motility ͉ cytoplasmic streaming ͉ molecular engineering C ytoplasmic streaming in characean algal cells is extremely fast, and this streaming is brought about by the movement of myosin-coated organelles along actin filament bundles fixed inside the cell (1). Myosin purified from Chara corallina could translocate actin filaments in the in vitro motility assay at a velocity comparable to that of the cytoplasmic streaming (approximately 50 m s Ϫ1 ) (2, 3). This velocity is about 10 times faster than that of the fast skeletal muscle myosin and the Chara myosin is the fastest motor protein known so far. We have cloned cDNA of the Chara myosin heavy chain (4) and succeeded in expressing functional motor domain (5, 6). The velocity of the expressed motor domain measured by in vitro motility assay was comparable to that of the native Chara myosin if we consider the difference in the lever arm length. Relevant steps in actomyosin ATPase cycle to the sliding velocity are ADP release from actomyosin and ATP reassociation resulting in actin-myosin dissociation. Kinetic analyses of Chara myosin motor domain revealed that both the ADP release and the ATP-induced dissociation from actin were very fast (6). Time spent in the strongly bound state with actin, which was estimated from these rates, was less than 1 ms. This very short strongly bound state, together with a large step size (7), are probably the reason for the very high velocity of Chara myosin. Actin-activated ATPase activity of expressed Chara motor domain was approximately 500 s Ϫ1 head Ϫ1

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“…We found that C-loop cleavage dramatically affects ␤S1 Mg 2ϩ -ATPase, suggesting that the C-loop participates in energy transduction (26). Actin binding protects Loop 2 from proteolysis in skeletal S1 indicating Loop 2 involvement in actin binding (30,31). Actin binding to ␤S1 fails to inhibit Loop 2 cleavage (26, 32) but does inhibit C-loop cleavage (26).…”

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confidence: 99%

The Myosin Cardiac Loop Participates Functionally in the Actomyosin Interaction

Ajtai

Garamszegi

Watanabe

et al. 2004

Journal of Biological Chemistry

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The motor protein myosin in association with actin transduces chemical free energy in ATP into work in the form of actin translation against an opposing force. Mediating the actomyosin interaction in myosin is an actin binding site distributed among several peptides on the myosin surface including surface loops contributing to affinity and actin regulation of myosin ATPase. A structured surface loop on ␤-cardiac myosin, the cardiac or C-loop, was recently demonstrated to affect myosin ATPase and was indirectly implicated in the actomyosin interaction. The C-loop is a conserved feature of all myosin isoforms with crystal structures, suggesting that it is an essential part of the core energy transduction machinery. It is shown here that proteolytic digestion of the C-loop in ␤-cardiac myosin eliminates actin-activated myosin ATPase and reduces actomyosin affinity in rigor more than 100-fold. Studies of C-loop function in smooth muscle myosin were also undertaken using sitedirected mutagenesis. Mutagenesis of a single charged residue in the C-loop of smooth muscle myosin alters actomyosin affinity and doubles myosin in vitro motility and actin-activated ATPase velocities, thereby involving a charged region of the loop in the actomyosin interaction. It appears likely that the C-loop is an essential electrostatic binding site for actin involved in modulation of actomyosin affinity and regulation of actomyosin ATPase velocity.The motor protein myosin is an ATPase and an actin-binding protein transducing ATP free energy into work. In muscle, myosin, actin, and ATP constitute the unitary work producer. The cyclical interaction of these components, a sequence of states each characterized as a static relation between the proteins and an intermediate in the degradation of ATP, is a contraction cycle. In a contraction cycle, myosin hydrolyzes ATP in its active site and forms a weak association with actin at a separate actin binding site. Release of phosphate from the active site initiates strong binding to actin and a large conformation change in myosin that produces work in the form of actin translation against a load. Several solved crystal structures represent intermediates in ATP hydrolysis and define myosin conformation changes in the absence of actin (1-6). Presently, we lack a detailed understanding of actomyosin structure and in particular how the elements making up the actomyosin interface contribute to mutual affinity, actin regulation of phosphate release, and the ability of myosin-bound nucleotide to modulate actomyosin affinity.Myosin consists of a globular head domain containing the enzymatic portion of the molecule and a tail involved in filament assembly. The globular head separated from its tail portion is called subfragment 1 (S1). 1 S1 interacts with filamentous actin (F-actin) consisting of polymerized actin monomers. An atomic model for F-actin built from monomeric actin crystal structures (7-9) satisfies x-ray diffraction data restraints (10) and is consistent with electron microscopic imagery (11). In...

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The limited tryptic cleavage of chymotryptic S-1 : An approach to the characterization of the actin site in myosin heads

Cited by 205 publications

References 23 publications

Effect of nucleotides, divalent cations and temperature on the tryptic susceptibility of myosin subfragment 1

Effect of nucleotides, divalent cations and temperature on the tryptic susceptibility of myosin subfragment 1

Unique charge distribution in surface loops confers high velocity on the fast motor protein Chara myosin

The Myosin Cardiac Loop Participates Functionally in the Actomyosin Interaction

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